Human adenoviruses are of major medical importance because they cause respiratory infections in children and young adults and because they are DNA tumor viruses. Fiber is a virus protein found at each vertex of the icosahedral capsid; it is important to a number of virus functions, including attachment to a cell surface receptor, assembly of infectious virions, and antigenicity. Fiber is thought to be a complex of three monomers, arranged into an N-terminal tail that binds to another capsid protein (penton base), an internal shaft, and a C-terminal knob that recognizes the cell surface receptor. Fiber from adenovirus serotypes 2 and 5 contains an unusual O-linked N-acetylglucosamine (O-GlcNAc), a saccharide modification found on many cytoplasmic and nuclear proteins (such as the proteins of the nuclear pore complex); O-GlcNAc may be important for a number of cellular functions, including assembly of multimeric complexes. These novel saccharides are added to fiber by a cytoplasmically oriented N-acetylglucosaminyltransferase shortly after synthesis of fiber molecules. Fibers from Ad2 and Ad5 also shared an epitope (recognized by antibody RL-2) with a nuclear pore protein (p62) and other O-GlcNAc-containing proteins. Fiber from Ad2 and Ad5 offers a unique system for the study of this O- GlcNAc modification and its potential roles in cellular processes, such as assembly of protein complexes. Ad2 and Ad5 fibers contain between 2 and 5 potential sites for O-GlcNAc attachment; amino acid sequencing of proteolytic fragments of fiber will be used to map the precise sites of attachment. A unique expression system, based on vaccinia virus recombinants, will be used to study mutants lacking one or more of the O- GlcNAc attachment sites. The proper assembly of fiber trimers can be monitored using a unique monoclonal antibody that recognizes only fiber trimers. Peptides containing the proposed sequences for O-GlcNAc attachment will be used to assay HeLa cell lysates for the N- acetylglucosaminyltransferase. Domains that encode the shared RL-2 epitope and that determine the ability to form trimers will be mapped. Domains in fiber that interact with penton base will also be mapped, using deletion and site-directed mutants made at the amino-terminus of fiber. A new form of cytoplasmic modification containing fucose and another carbohydrate have recently been detected by labeling of hexon and penton base. As a long term goal, this novel modification will be identified and its site of attachment mapped, using techniques similar to those described above for the study of O-GlcNAc. These studies may lead to the elucidation of the role of this newly detected modification on adenovirus nd other cellular proteins.